The present invention relates to the immunodeficiency virus SIM27 of drill monkeys, whose RNA or a part thereof is complementary to the sequence shown below, and variants of this virus. Moreover, the viral RNA, the corresponding CDNA, proteins derived therefrom and fragments of the nucleic acids or proteins are a subject of the present invention. The invention likewise relates to the diagnostic use of the mentioned nucleic acids and proteins and their fragments, and a diagnostic comprising these nucleic acids and/or proteins and/or fragments thereof.
Primates have been developing for approximately 30 million years, which has lead to a high degree of variability of the individual primate species. The New World monkeys (Platyrrhini) are differentiated from the Old World monkeys (Catarrhini), which for their part are divided into the hominoids (Hominoidae) and the cercopithecoids (Cercopithecoidea). Together with the primates, various infective agents have also developed, which have adapted to the individual primate species or, for example, to a whole family. Examples of virus are the simian pathogenic and the human pathogenic herpesviruses, which although they can still infect individuals of another primate species, are naturally not transmitted from one primate species to the other. Other viruses still infect all primates, such as the rabies virus, the yellow fever virus and the filovirus.
Lentiviruses are subdivided into the genera of the spume viruses, the T-leukemia/lymphoma viruses and the immunodeficiency viruses. A general survey of the leukemia and immunodeficiency viruses of the monkeys and their pathogenicity is found in the article of Hayami (Hayami M et al., Curr. Top. Microbiol. Immunol. 1994; 188: 1-20). Spume viruses appear to occur only in monkeys. Since until now a pathogenicity of the spume viruses has not been detected, this virus is being less intensively investigated than HIV/SIV and HTLV/STLV.
HTLVs, the human T-leukemia viruses type I and type II, are structurally very similar to STLVs, the simian (monkey) T-lymphoma viruses (Franchini et al., AIDS Res Human Retrovirus 1994; 10: 1047-1060). Thus the difference in the virus species, STLV I and II, and the viruses between man (HTLV) and monkeys (STLV) is a sign of a long individual evolution in the individual primates, if a cross-transmission between the various primate species can be excluded (Franchini et al., AIDS Res Human Retrovirus 1994; 10: 1047-1060). STLV-infected monkeys occur over the entire world (Hayami M et al., Curr. Top. Microbiol. Immunol. 1994; 188: 1-20), whereas SIV-infected monkeys are only to be found naturally in Africa, which is an indication of the fact that SIV very probably developed later than STLV.
Molecular biology results show clearly that HIV-1 is very closely related to the immunodeficiency viruses of the chimpanzee. The latter viruses are subsequently designated as SIV-1, whereas the virus of the mangabeys, SIVsm, is designated as SIV-2. SIV-1 and HIV-1 derive with high probability from a precursor virus, just as SIV-2 and HIV-2 probably have a common precursor. Up to 25% of troops monkeys can naturally be infected with SIV-2 without signs of the virus pathogenesis being detectable in the infected animals (Chen Z et al., J Virol. 1996; 70: 3617-3627). In the case of SIV-2, infections in man were detected which do not differ in their pathogenesis from an HIV-2 infection. SIV-2 is closely related to HIV2 and particularly epidemically widespread in West Africa south of the Sahara, in the same region in which the mangabeys live (Gao FL et al., Nature 1992; 358: 495-499). The results of the investigations on SIV show that in addition to the SIV-2 (SIVsm) of mangabeys the immunodeficiency viruses of the African green meerkat represent a further type, perhaps SIV-3, and in addition meanwhile some further simian SIVs have been isolated which cannot be assigned to the groups of viruses mentioned and which probably represent the SIV type 4. This SIV-4 type is formed by the viruses of the Sykes monkeys (Cercopithecus mitis), the Hoest monkeys (Cercopithecus 1xe2x80x2hoesti) (Hirsch VM et al., J. Virol. 1999; 73: 1036-1045), the red cap mangabeys (Cercopithecus torquatus torquatus) (Georges-Courbot MC et al., J. Virol. 1998; 72: 600-608), the mandrill monkeys SIVmnd (Mandrillus sphinx) (Tsujimoto H et al., Nature 1989; 341: 539-541), and the drill monkeys (Mandrillus leucophaeus) (Clewley JP et al., J. Virol. 1998; 72: 10305-10309). All previously isolated SIV-4s can be cultured in human peripheral blood lymphocytes and some in the human permanent cell line Molt4 clone 8 (Hirsch VM et al., J. Virol. 1999; 73: 1036-1045), which indicates that the infection of man with these viruses should also be possible. The SIV-4 type is so different from the SIV-2 type that an SIVmac(SIV2)-specific p25 antigen test cannot detect SIVhoest (SIV4) produced in the supernatant of infected cells (Hirsch VM et al., J. Virol. 1999; 73: 1036-1045), as the Gag region is too divergent for recognition by monoclonal antibodies. The phylogenetic comparison of the nucleic acid sequences of the simian viruses also shows that the SIV-4 described here differs from SIV-2 and SIV-3 (Korber et al. Human Retroviruses and AIDS 1997. A compilation and analysis of nucleic acid and amino acid sequences. Los Alamos National Laboratory, New Mexico, 1998).
As described above, a virus similar to SIVcpz is possibly the precursor virus of viruses causing human HIV-1 infections, which the high similarity of viruses of the group HIV1-M, xe2x88x92N and xe2x88x92O to SIV-1 indicates.
To date, there are no reports that humans have been infected with SIV-4. A nosocomial infection with SIV-3 or SIV-2 occurred due to contamination of the eczematous skin of a laboratory assistant (Khabbaz RF et al., N. Engl. J. Med. 1994; 330: 172-177). The SIV replicated for a certain time which was sufficient for the induction of a strong antibody response, but was not sufficient to establish a permanent infection (Khabbaz RF et al., N. Engl. J. Med. 1994; 330: 172-177). About 3.5 years after seroconversion, the laboratory assistant appeared to be free of the infection (Khabbaz RF et al., N. Engl. J. Med. 1994; 330: 172-177). Whether this path of virus elimination is the rule or whether persistent infections with corresponding pathogenesis can also result from the infective event is unknown.
Since until now no epidemiological studies on target groups in central Africa have been carried out which can show whether variant viruses such as SIV-4 also circulate in the human population, infection of man cannot be confirmed, but can also not be excluded.
As was seen in the example of the HIV-1 subtype O, antibody detection tests on the basis of HIV-1 subtype M were not sufficiently reactive in order to be able to detect all subtype O-infected patients (Simon F et al. AIDS 1994; 8: 1628-1629). The diagnosis of an infection with an aberrant human pathogenic SIV subtype could probably also not be made, as it must be assumed that the ELISA exploratory tests based on HIV-1 and/or HIV-2 antigens are negative or would only be slightly reactive, and the attempt at confirmation by means of the immunoblot produced a negative or probably questionable result. The diagnosis could probably also not be made by means of the nucleic acid tests, since with the presently available tests, for example, neither the nucleic acid of the viruses of group O nor that of HIV-2 can be reliably amplified (Gxc3xcrtler L et al., 12th World AIDS Conference Geneva Basic Science 1: 121-124).
The drill monkeys described here (Mandrillus leucophaeus) are animals which originate from the western region of Cameroon bordering Nigeria and live wild there in the bushland. Drill monkeys have become widespread in the central West-African region. The animals are hunted and eaten, which is why the stock in recent years has continuously decreased. Young animals are in some cases picked up and kept in the vicinity of the houses as pets. The monkey 27 described here (3 years old) was captured from a free hunting reserve and then domesticated over the course of a year and has had no contact with other monkeys of the same or of a similar species.
As described in Example 2, the virus originating from monkey 27 was replicated in human PBLs. Genomic DNA and thus also integrated proviral DNA of the SIV was isolated from the infected cells. The deciphering of the sequence of the total genome of the SIV is described in Example 3. The PCR (polymerase chain reaction) method was employed for the multiplication of the viral DNA. The components needed for carrying out the process can be acquired commercially.
Using this process, it is possible to amplify DNA sequences if DNA regions of the sequence to be amplified are known, or known sections are sufficiently similar. Short complementary DNA fragments (oligonucleotides=primers) which add to a short region of the nucleic acid sequence to be amplified must then be synthesized. For carrying out the test, nucleic acids are combined with the primers in a reaction mixture which additionally contains a polymerase and nucleotide triphosphates. The polymerization (DNA synthesis) is carried out for a specific time, then the nucleic acid strands are separated by warming. After cooling, the polymerization starts again.
The amplified genome sections were sequenced by the Sanger method. As described in Example 4, the genome of SIM27 was subjected to phylogenetic comparisons which showed that it is a strongly divergent novel simian immunodeficiency virus.
The present invention therefore relates to:
1.) Immunodeficiency viruses which branch off as a side branch from the SIM27 side branch after the branching of SIM27 in a phylogenetic investigation of their total genome on the nucleic acid plane, as is described in Example 4 (see FIG. 1)
2.) GAG proteins and fragments thereof which branch off as a side branch from the SIM27 side branch after the branching of SIM27 in a phylogenetic investigation of their total sequence on the amino acid plane, as is described in Example 4 (see FIG. 2).
3.) Pol proteins and fragments thereof which branch off as a side branch from the SIM27 side branch after the branching of SIM27 in a phylogenetic investigation of their total sequence on the amino acid plane, as is described in Example 4 (see FIG. 4), or a POL protein fragment or subfragments thereof which branch off as a side branch from the SIM27 side branch after the branching of SIM27 in the region of the sequence including this amino acid sequence, published by Clewley (Clewley JP et al., J. Virol. 1998; 72: 10305-10309), as has been investigated as described in Example 4 (see FIG. 6).
4.) ENV proteins and fragments thereof which branch off as a side branch from the SIM27 side branch after the branching of SIM27 in a phylogenetic investigation of their total sequence on the amino acid plane, as is described in Example 4 (see FIG. 7).
Of particular interest is furthermore the consideration of the strongly immunogenic cysteine loop region in the Erxv gene, which is therefore of particular diagnostic importance. The cysteine-loop regions of various immunodeficiency viruses are shown in Table 1 (SEQ ID NOS: 26-57, respectively).
As can be clearly seen, either lysine or arginine occurs in position 3 of the cysteine loop (C12345C) in nearly all representatives of immunodeficiency viruses. The only exception up to now was found in the immunodeficiency virus MNDGB1, which was likewise isolated from a drill monkey (Mandrillus spinx). With great probability it is to be assumed from this that antibodies formed against this modified epitope cannot be recognized or can be recognized with clearly decreased efficiency from diagnostic tests known up to now which are based on the customary arginine- or lysine-containing antigens.
This invention therefore likewise relates to antigens in which arginine and/or lysine within the cysteine loop region in position 3 has been replaced by any desired amino acid, particularly preferably a polar amino acid such as serine or an amino acid having an aliphatic side chain such as alanine.
The present invention is moreover described in the examples and in the patent claims, where the examples serve for summarization and no restriction of the present invention must be derived therefrom.
Identification of the SIM27 infection in drill monkeys
In the course of a study, EDTA blood was taken from drill monkeys in the villages of rural Cameroon, in which they were kept, and this was analyzed in various HIV tests. On testing the serum of the monkey SIM27 for antibodies, a competitive ELISA for HIV-1 was negative and an ELISA from Dade Behring (Enzygnost HIV-1/2 plus) recognizing HIV-1, xe2x88x922 and xe2x88x92O was likewise negative, the extinction lying near the threshold value. In the analysis of the HIV-1 Western blot (virus MVP899-87) which was carried out at the same time, no virus-specific bands were to be seen, in the HIV-2 blot (virus MVP11971-87), the band gp36 was to be seen strongly, and the bands p55 and p68 were to be seen, and in the HIV-1 group O blot (virus MVP5180-91), the bands p24 and p55 were to be seen. Gp36 is the transmembrane protein of HIV-2, the bands p55 and p68 correspond to the reverse transcriptase (p55) plus the RNaseH (p68) of HIV-2, and p24 is the inner core protein of HIV-1 group O viruses and p55 the precursor protein of gag and thus also p24. 20 ml of plasma from the animals were employed in order to develop the Western blot. According to the analysis of the nucleic acid sequence, the virus MVP11971-87 is a representative of the group HIV-2A, the virus MVP899 a representative of HIV-1B.
The SIV infection of the monkeys with the drill virus is thus distinguished:
by negativity in normal screening ELISAs for HIV antibodies,
by serological cross reaction in the env and pol region with the HIV-2 transmembrane glycoprotein and the reverse transcriptase in the Western blot,
by serological cross reaction in the gag region with the inner core protein of HIV-1 group O and absent cross reaction with the core proteins of group M (HIV-1B) in the Western blot.
The lymphocyte fraction was isolated by Ficoll gradient centrifugation from 5 ml each of EDTA blood of the monkeys. The lymphocytes were stimulated with PHA (phytohemaglutinih, 5 mg/ml) and PMA (myristylphorbol ester, 10 ng/ml), after 3 days both additives were washed out and the culture was continued in the presence of RPMI-1640, as usual, with interleukin-2 addition. The PMA stimulation was described by Kubo et al. (Kubo M et al., J. Virol 1997; 71: 7560-7566).
The culture conditions were similar to those which have been described by Tamalet et al. (Tamalet, C. et al., AIDS 1994; 1083-1088). After one week in culture, human PHA-stimulated and nonstimulated blood lymphocytes (PBLs) were added to the monkey lymphocytes and the addition was repeated once weekly until it was possible after about 3 weeks to detect beginning SIV production by means of a commercially obtainable p24 antigen test (Abbott, Wiesbaden).
The virus was then subcultured on human lymphocytes from the supernatant of the cells. All attempts to transfer the SIM27 to permanent culture cells such as HUT-78 or Jurkat have failed up to now. By means of monthly subculturing, it was possible to keep SIM27 on PBL in culture for 9 months from then on.
Genomic DNA from SIM27-infected blood lymphocytes was isolated by standard methods (Current Protocols in Molecular Biology, Wiley Interscience, 1994).
The total genome was amplified exclusively by means of PCR (polymerase chain reaction). All PCRs were begun by means of xe2x80x9cHot Startxe2x80x9d: after addition of all components of the PCR, except the polymerase, this was added only after heating the sample to 94xc2x0 C., which strongly reduces the extension of nonspecifically binding primers.
A general survey of the individual stages of the deciphering of the genome is shown in FIG. 8.
For the characterization of genome regions of the isolate SIM27, PCR experiments were carried out with primer pairs from the region of the integrase in the pol gene. The PCR (Saiki et al., Science 239: 487-491, 1988) was modified as follows:
For the first amplification of HIV-specific DNA regions, 5 xcexcl (200 xcexcg/ml) of genomic DNA from SIM27-infected blood lymphocytes were pipetted into a 50 xcexcl reaction mixture (0.25 mM dNTP, 1 xcexcM each primer, 10 mM tris HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 2.5 units platinum-Taq DNA polymerase (Gibco)) and amplified according to the following temperature program:
1) initial denaturation: 3 min. 95xc2x0 C.,
2) amplification: 30 sec. 94xc2x0 C., 30 sec. 49xc2x0 C., 30 sec. 68xc2x0 C. (30 cycles).
The primers used for the PCR had the following sequence:
(Seq. ID No. 1 and 2)
5xe2x80x23xe2x80x2
5pol2380agm GCC ATG TGT CCA AAA TGT CA
3pol2930agm CTT CTC TGT AGT AGA CTC TA
5 xcexcl of the amplificate were employed as a template for a second nested PCR with the following primers and the same temperature profile:
(Seq. ID No. 3 and 4)
5pol2460agm TAG TAG CAG TCC MYR KWG
(M=A/C,Y=C/T,R=A/G,K=G/T,W=A/T)
3pol2760agm TCT CTA ATT TGT CCT ATG AT
The amplificate thus obtained was sequenced directly without cloning.
The sequence found is shown in Table 2.
Based on the publication of Clewley (Clewley JP et al., J. Virol 1998; 72: 10305-10309), a further amplificate was obtained in the 5xe2x80x2 region of the pol gene. The primers DR1, DR2 and, for the nested PCR, DR4 and DR5 described by Clewley were used, as well as the temperature cycles described in this publication. The polymerases used were DNA-Taq polymerase (Perkin Elmer) and the buffers described above.
The sequence according to Table 3 was obtained here:
In a next amplification, the region of SIM27 lying between the amplificates already obtained was amplified. The primers mentioned below were used here.
For the first PCR:
(Seq. ID No. 5 and 6)
1216 ATG CCC ATT GGA TGA GGA C
1197 GAC TGT GGC TAC CTT TTC ACT
For the nested PCR:
(Seq. ID No. 7 and 8)
1218 CAT CGG TGA ATA ATC AGG
1226 GGT ATT ACT TCT GCC TCT A
The platinum-Taq DNA polymerase (Gibco) was used according to the following temperature program:
1) initial denaturation: 2 min. 95xc2x0 C.,
2) amplification: 30 sec. 95xc2x0 C., 30 sec. 55xc2x0 C., 150 sec. 68xc2x0 C. (30 cycles).
The sequence according to Table 4 was obtained here.
The region of the total sequence of the 5xe2x80x2-LTR region of the genome up to the pol gene was amplified with the following primer pairs:
1. PCR:
(Seq. ID No. 9 and 10)
1248 CTC AAT AAA GCT TGC CTT GA
1217 GTC CTC ATC CAA TGG GCA T
2. Nested PCR:
10 (Seq. ID No. 11 and 12)
1249 TRD CTA GAG ATC CCT CAG A (R=A/, D=G/A/T)
1219 CCA ATA CTG TGA TCT GTT CAC
platinum-Taq DNA polymerase (Gibco) was in each used according to the following temperature program:
1) initial denaturation: 2 min. 95xc2x0 C.,
2) amplification: 30 sec. 95xc2x0 C., 30 sec. 50xc2x0 C., 180 sec. 68xc2x0 C. (30 cycles). 1xc3x97enhancer (Gibco) was used in addition to the buffers indicated above.
The sequence according to Table 5 was obtained here:
The still missing region of the total sequence of the integrase up to the 3xe2x80x2-LTR was amplified with the following primer pairs, the primer 1270 being discarded on account of the sequence of the 5xe2x80x2 LTR region (prior amplificate):
1. PCR:
(Seq. ID No. 13 and 14)
1246 CCT ATT CAT GGC CAG GTA
1270 GAT TTT TCT CTA CTC TCA CTA
2. Nested PCR:
(Seq. ID No. 15 and 16)
1196 AGT GAA AAG GTA GCC ACA GTC
12710 GAT TTT TCT CTA CTC TCA CTA
The platinum-Taq DNA polymerase (Gibco) was in each case used according to the following temperature program:
1) initial denaturation: 2 min. 95xc2x0 C.,
2) amplification: 30 sec. 95xc2x0 C., 30 sec. (47xc2x0 C. 1.PCR; 51xc2x0 C. 2. PCR), 360 sec. 68xc2x0 C. (30 cycles). 1xc3x97enhancer (Gibco) was used in addition to the buffers indicated above.
The sequence according to Table 6 was obtained here:
The total sequence which results from the sum of the sequences according to Tables 2 to 6 is shown in Table 7:
In 3 reading frames, the nucleotide sequence was converted into amino acid sequences, after which the amino acid sequences of GAG (Table 8), POL (Table 9) and ENV (Table 10) were identified by homology comparisons.
Selection of the sequences:
From the HIV WWW server of the LANL (Los Alamos National Laboratory, hiv-web.lanl.gov), 31 HIV and SIV sequences were selected which all comprised complete SIV genomes and representatives of the various HIV-1 and HIV-2 subtypes. The following sequences according to Table 11 were taken into consideration.
With the aid of the Genbank accession numbers of these sequences, the actual sequence entries were extracted from the gene database xe2x80x9cGenbankxe2x80x9d. With the aid of annotation, the genes env, gag and pol were extracted from these sequences and translated into the amino acid sequence. For the translation, only those sequences were used which were annotated as functional. Pseudogenes and genome sections not annotated as one of the 3 genes were not taken into consideration.
In addition, the sequence of the genome of SIM27 was compared with the actual gene database xe2x80x9cGenbankxe2x80x9d in order not to overlook an SIV partial sequence having a high relationship to SIM27. 2 partial sequences of SIVrcm (gag and pol) and a pol partial sequence of Mandrillus leucophaeus (Clewley JP et al., J. Virol. 1998; 72: 10305-10309) were identified as additionally relevant here:
In total, 4 data sets were obtained in this way: 3 protein data sets (env, gag and pol), and one from genomic sequences (GENOME).
Alignment:
The above sequences were aligned together with the corresponding SIM27 sequences using CLUSTALW (Version 1.74) with standard settings (Thompson J. D et al., Nucleic Acids Res. 22: 4673-4680 (1994)). The sequence alignments thus obtained were then checked manually.
The published pol partial sequence of drill monkeys (Clewley et al.), and the pol partial sequence of the RCM monkey was added once more each to the pol sequence alignment in analyses which were separate in each case. The same was carried out for the GAG partial sequence of the RCM monkeys for the gag alignment.
For the addition of the individual sequences to the alignments, the profile alignment option of CLUSTALW 1.74 was used with standard settings.
3 further protein data sets with small partial sequences RCM-GAG, RCM-POL and DRILL-POL thus resulted. Each of these data sets was considered only with respect to the region of the respective partial sequence.
Phylogenetic Analyses
Using the above seven alignments (GENOME (FIG. 1), GAG (FIG. 2), RCM-GAG (FIG. 3), POL (FIG. 4), RCM-POL (FIG. 5), DRILL-POL (FIG. 6), ENV (FIG. 7)), phylogenetic family trees were then independently set up. For this, the neighbor-joining method, as is implemented in CLUSTALW 1.74, was used in 1000 boot strap analyses. To calculate the trees, the standard settings were used, only all alignment gaps with holes were ignored, and the correction for multiple mutations was switched on.
According to known methods of molecular biology (Current Protocols in Molecular Biology, Wiley Interscience, 1994), the region of env containing the cysteine loop was stabily expressed either as a fusion with the maltose-binding protein (pMAL-New England Biolabs) or as a fusion with xcex2-Gal (Knapp et al., Biotechniques, Vol. 8, No. 3, 1990). The proteins were blotted on nitrocellulose, incubated overnight with the sera in a dilution of 1:100 in TBS containing 5% skimmed milk (150 mM NaCl, 50 mM tris pH 8.0), washed with TBS and incubated with anti-human IgG-AP (Sigma A064) and anti-monkey IgG-AP (Sigma A1929) for 2 h in a dilution of 1:1000 and stained according to the manufacturer""s instructions by means of Nitrotetrazolium Blue (Sigma N-6878) and 5-bromo-4-chloroindolyl phosphate.p-toluidine (Bachem M105). The results shown in Table 9 were obtained (FIG. 9).
It was surprisingly seen here that the env region of SIM27 does not react with anti-HIV-1, anti-HIV-1 subtype O and anti-HIV2 sera and at the same time antibodies from SIM27, which react strongly with SIM27-env, could not be detected by the use of HIV-1-env, HIV-1-subtype O env and HIV2-env. It is therefore to be assumed from this that in the case in which SIM27 or a variant with comparable serological properties ought to complete the transition into the human population, the detection of antibodies against SIM27 in human sera is not possible with the tests currently employed, but rather SIM27-env, or antigens derived therefrom having comparable immunological properties, have to be employed.